The experiment was repeated twice. To validate the interaction data by an independent approach, we selected some of the VipA mutants and tested them for binding to VipB in the Y2H click here system using two independent reporter genes: lacZ, which allows us to compare the relative strength of the VipA-VipB interactions by quantification of β-galactosidase activity, and MEL1, which in the case of a positive interaction and in the presence of the substrate X-α-Gal will promote blue color development. According to both reporters, the deletion mutant Δ104-113, the double

mutant V110A/L113A and the quadruple mutant D104A/V106A/V110A/L113A were all essentially unable to bind VipB and produced α-and β-galactosidase levels similar to the negative vector control, while the double mutant D104A/V106A and the triple mutant D104A/V106A/V110A

both showed intermediate binding (Table 1 and data not shown). The less sensitive MEL1 reporter KPT-330 research buy assay did not detect any obvious binding defects for single mutants D104A, V106A or V110A (data not shown), while the lacZ reporter revealed a weak binding defect for both V106A and V110A mutants (Table 1). Thus, overall, the Y2H data confirms the results from the E. coli B2H assay. Table 1 Protein-protein interactions in the yeast two-hybrid assay DNA-binding domain Activation domain Relative β-gal activity VipB None 0.5 ± 0.1% *** VipB VipA 100.0 ± 5.8% VipB VipA Δ104-113 1.0 ± 0.2% *** VipB VipA D104A 92.7 ± 4.1% VipB VipA V106A 92.4 buy LXH254 ± 3.4% * VipB

VipA V110A 74.6 ± 3.4% *** VipB VipA D104A/V106A 64.1 ± 10.7% * VipB VipA V110A/L113A 1.1 ± 0.3% *** VipB VipA D104A/V106A/V110A 48.8 ± 2.0% *** VipB VipA D104A/V106A/V110A/L113A 1.0 ± 0.2% *** VipA mutants fused to the GAL4 activation domain of plasmid pGADT7 were co-transformed with VipB on the GAL4 DNA-binding domain pGBKT7 into the S. cerevisiae reporter strain Y187. Activation of the lacZ reporter from 4 independent experiments where duplicate transformants were tested on each occasion was determined and expressed as % mean β-galactosidase activity ± SEM relative to the activity of the wild-type protein. A Student’s 2-sided t-test was used to determine whether the differences observed were statistically significant (*, P < 0.05; ***, P < 0.001). Recently, we have shown that temperature and salinity influences the activity of the T6SS of V. cholerae O1 strain Lonafarnib A1552 [13]. To determine whether salt and/or temperature also influence(s) the interaction of VipA and VipB, we compared the strength of the interaction in the B2H assay when E. coli was grown under different salt and temperature conditions. The results suggest that E. coli grown in Luria Broth (LB) supplemented with additional NaCl (high salt) over night, generally produce higher β-galactosidase activity than if grown in low salt (i.e. normal LB) (Figure 3). This suggests that a high concentration of salt is beneficial for the VipA-VipB interaction.